The broad goals of this proposal are to provide a molecular-level understanding of how RNA enzymes (ribozymes) catalyze chemical reactions. We are studying a self-cleaving RNA that was originally identified in the human hepatitis delta virus, but is now known to be widely distributed in nature. This ribozyme harnesses a nucleobase with a dramatically shifted pKA and a divalent metal ion to catalyze an RNA cleavage reaction. We will integrate X-ray crystallography, molecular dynamics, and solution biochemistry experiments to learn how the three dimensional structure of the RNA interacts with its metal cofactors to achieve catalysis and how the molecular motions of this dynamic RNA contribute to its reactivity. Our first specific aim describes the strategies we will use to solve the three dimensional structure using X-ray crystallography, and to verify that the conformation and any disorder observed in the crystal mimics the conformation of the active ribozyme in solution. In our second specific aim, we will use molecular dynamics to characterize the motions that occur within the ribozyme active site and to understand the role of disorder in ribozyme catalysis. The last specific aim describes biochemical and spectroscopic experiments designed to dissect the contributions of active site components to catalysis. We will analyze potential ligands to the catalytic metal ion, probe the mechanism by which the catalytic metal ion contributes to catalysis, and explore using solution biochemistry the positioning and motions of nucleotides upstream of the scissile phosphate and how they contribute to the reaction pathway. The results of this study will provide an in-depth structural and mechanistic analysis of one ribozyme. However, in the post genomic age, we are seeing an unexpected contribution of non-coding RNA sequences to regulation of gene expression. Ribozymes and riboswitches are being discovered in a variety of contexts, including within eukaryotic transcriptomes. It is therefore essential to have in our knowledge base some in-depth knowledge of a few paradigm systems such as the hepatitis delta virus ribozyme in order to fully understand the catalytic potential of common ribozymes and unique orphan ribozymes. PUBLIC HEALTH RELEVANCE: In the post genomic age, we are seeing an unexpected contribution of non-coding RNA sequences, including ribozymes, to regulation of prokaryotic and eukaryotic gene expression. To fully understand how these non-coding, functional RNAs work, we are undertaking an in depth structural and mechanistic analysis of a ribozyme. We anticipate that the results of this study will provide clues as to the catalytic strategies of many ribozymes, some of which will be therapeutic targets.